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1.
Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation 总被引:2,自引:2,他引:0
Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 n M ) and okadaic acid (50 n M ) reduced significantly ( p < 0.01) the synthesis of [3 H]ACh from [3 H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner ( p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly ( p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner ( p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly ( p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [3 H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced V max values for high- and low-affinity choline uptake ( p < 0.01) with no significant change in K m values ( p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis. 相似文献
2.
Jesús Manuel de la Fuente Amalia Vázquez M. Mar González Miguel Sánchez María Molina César Nombela 《Applied microbiology and biotechnology》1993,38(6):763-769
Thermosensitive mutant strains of Saccharomyces cerevisiae that fail to generate an osmotically stable cell wall when grown at a non-permissive temperature release their cell contents upon expression of the mutation. Therefore, they may represent an alternative for the production of homologous or heterologous protein preparations. In order to analyse the expression of two of these mutations, lyt2 and slt2, we grew the corresponding strains under precisely defined conditions in batch and continuous fermentors. A switch in the temperature of batch cultures from 24° C to 37° C determined lysis of the cells with a significant release of intracellular enzymes. These include alkaline phosphatase and periplasmic proteins such as glucan-degrading enzymes, the pattern of cell lysis and protein release being maintained for about 6 h. One-stage continuous cultures of a lyt2 mutant were maintained for long periods at 37° C; a fraction of the population lysed and released the indicated proteins, but eventually a revertant of the lytic phenotype was selected. To avoid this, a two-stage continuous culture system was developed by connecting two fermentors in series, the effluent from the first one at 24°C being fed to the second one adjusted to 37° C. A steady state of cell lysis and protein liberation was reached in the second-stage fermentor without any evidence of selection of revertants. This system can be very useful for developing conditions for the use of yeast strains to produce protein preparations.
Correspondence to: C. Nombela 相似文献
3.
4.
J. del Mazo J. A. Abrisqueta Amalia Pérez-Castillo V. Aller M. Angeles Martín Lucas M. Luisa de Torres M. José Martín 《Human genetics》1978,44(1):105-108
Summary A new case of ring chromosome 4 in a 2-day-old female child with multiple malformations is described. By means of the GTG-banding technique, a karyotype 46,XX,r(4), (p16q35) was determined. The characteristics of the child's karyotype and the relationship with the structure of the chromosome, especially the location of the deletion that produces the syndrome, are compared with previous reports. 相似文献
5.
The NHERF1 PDZ2 domain regulates PKA-RhoA-p38-mediated NHE1 activation and invasion in breast tumor cells 总被引:1,自引:0,他引:1 下载免费PDF全文
Cardone RA Bellizzi A Busco G Weinman EJ Dell'Aquila ME Casavola V Azzariti A Mangia A Paradiso A Reshkin SJ 《Molecular biology of the cell》2007,18(5):1768-1780
Understanding the signal transduction systems governing invasion is fundamental for the design of therapeutic strategies against metastasis. Na(+)/H(+) exchanger regulatory factor (NHERF1) is a postsynaptic density 95/disc-large/zona occludens (PDZ) domain-containing protein that recruits membrane receptors/transporters and cytoplasmic signaling proteins into functional complexes. NHERF1 expression is altered in breast cancer, but its effective role in mammary carcinogenesis remains undefined. We report here that NHERF1 overexpression in human breast tumor biopsies is associated with metastatic progression, poor prognosis, and hypoxia-inducible factor-1alpha expression. In cultured tumor cells, hypoxia and serum deprivation increase NHERF1 expression, promote the formation of leading-edge pseudopodia, and redistribute NHERF1 to these pseudopodia. This pseudopodial localization of NHERF1 was verified in breast biopsies and in three-dimensional Matrigel culture. Furthermore, serum deprivation and hypoxia stimulate the Na(+)/H(+) exchanger, invasion, and activate a protein kinase A (PKA)-gated RhoA/p38 invasion signal module. Significantly, NHERF1 overexpression was sufficient to induce these morphological and functional changes, and it potentiated their induction by serum deprivation. Functional experiments with truncated and binding groove-mutated PDZ domain constructs demonstrated that NHERF1 regulates these processes through its PDZ2 domain. We conclude that NHERF1 overexpression enhances the invasive phenotype in breast cancer cells, both alone and in synergy with exposure to the tumor microenvironment, via the coordination of PKA-gated RhoA/p38 signaling. 相似文献
6.
The NMR determination of the structure of Cd(7)-metallothionein was done previously using a relatively large protein concentration that favors dimer formation. The reactivity of the protein is also affected under this condition. To examine the influence of protein concentration on metallothionein conformation, the isolated Cd(4)-alpha-domain was prepared from rabbit metallothionein-2 (MT 2), and its three-dimensional structure was determined by heteronuclear, (1)H-(111)Cd, and homonuclear, (1)H-(1)H NMR, correlation experiments. The three-dimensional structure was refined using distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. The backbone superposition of the alpha-domain from rabbit holoprotein Cd(7)-MT 2 and the isolated rabbit Cd(4)-alpha was measured at a RMSD of 2.0 A. Nevertheless, the conformations of the two Cd-thiolate clusters were distinctly different at two of the cadmium centers. In addition, solvent access to the sulfhydryl ligands of the isolated Cd(4)-alpha cluster was 130% larger due to this small change in cluster geometry. To probe whether these differences were an artifact of the structure calculation, the Cd(4)-alpha-domain structure in rabbit Cd(7)-MT 2 was redetermined, using the previously defined set of NOEs and the present calculation protocol. All calculations employed the same ionic radius for Cd(2+) and same cadmium-thiolate bond distance. The newly calculated structure matched the original with an RMSD of 1.24 A. It is hypothesized that differences in the two alpha-domain structures result from a perturbation of the holoprotein structure because of head-to-tail dimerization under the conditions of the NMR experiments. 相似文献
7.
Annalisa Cossu Anna Maria Posadino Roberta Giordo Costanza Emanueli Anna Maria Sanguinetti Amalia Piscopo Marco Poiana Giampiero Capobianco Antonio Piga Gianfranco Pintus 《PloS one》2012,7(11)
The cardiovascular benefits associated with diets rich in fruit and vegetables are thought to be due to phytochemicals contained in fresh plant material. However, whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed apricots were isolated and their presence confirmed by colorimetric analysis and browning index. Oxidative injury of endothelial cells (ECs) is the key step for the onset and progression of cardiovascular diseases (CVD), therefore the potential protective effect of apricot melanoidins on hydrogen peroxide-induced oxidative mitochondrial damage and cell death was explored in human ECs. The redox state of cytoplasmic and mitochondrial compartments was detected by using the redox-sensitive, fluorescent protein (roGFP), while the mitochondrial membrane potential (MMP) was assessed with the fluorescent dye, JC-1. ECs exposure to hydrogen peroxide, dose-dependently induced mitochondrial and cytoplasmic oxidation. Additionally detected hydrogen peroxide-induced phenomena were MMP dissipation and ECs death. Pretreatment of ECs with apricot melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide-induced intracellular oxidation, mitochondrial depolarization and cell death. In this regard, our current results clearly indicate that melanoidins derived from heat-processed apricots, protect human ECs against oxidative stress. 相似文献
8.
Cortés-Sarabia Karen Bautista-Santos Isabel Cisneros-Sarabia Alondra Parra-Rojas Isela Estrada-Moreno Ana Karen Flores-Alfaro Eugenia Fernández-Tilapa Gloria Ramírez-Peralta Arturo Vences-Velázquez Amalia 《International journal of peptide research and therapeutics》2021,27(1):413-420
International Journal of Peptide Research and Therapeutics - Gardnerella vaginalis produces vaginolysin (VLY), a cholesterol-dependent cytolysin, responsible of the cellular lysis and epithelial... 相似文献
9.
In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4–7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively. 相似文献
10.
Shevin T. Jacob Patricia B. Pavlinac Lydia Nakiyingi Patrick Banura Jared M. Baeten Karen Morgan Amalia Magaret Yuka Manabe Steven J. Reynolds W. Conrad Liles Anna Wald Moses L. Joloba Harriet Mayanja-Kizza W. Michael Scheld 《PloS one》2013,8(8)